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- EMDB-13486: Cryo-EM structure of ShCas12k in complex with a sgRNA and a dsDNA... -

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Basic information

Entry
Database: EMDB / ID: EMD-13486
TitleCryo-EM structure of ShCas12k in complex with a sgRNA and a dsDNA target
Map data
Sample
  • Complex: Ternary complex of ShCas12k with sgRNA and target dsDNA
    • Protein or peptide: ShCas12k
    • RNA: sgRNA
    • DNA: DNA target strand
    • DNA: DNA non-target strand
KeywordsCas12k / sgRNA / target DNA / Protein-RNA-DNA complex / Tn7 / DNA Binding Protein / Transposition / CRISPR
Function / homology: / ShCas12k
Function and homology information
Biological speciesScytonema hofmannii (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.04 Å
AuthorsSchmitz M / Jinek M
Funding supportEuropean Union, 2 items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_182567European Union
European Research Council (ERC)ERC-CoG-820152European Union
CitationJournal: Nature / Year: 2021
Title: Target site selection and remodelling by type V CRISPR-transposon systems.
Authors: Irma Querques / Michael Schmitz / Seraina Oberli / Christelle Chanez / Martin Jinek /
Abstract: Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided ...Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion. Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ, but the molecular mechanism of RNA-directed DNA transposition has remained elusive. Here we report cryo-electron microscopic structures of a Cas12k-guide RNA-target DNA complex and a DNA-bound, polymeric TnsC filament from the CRISPR-associated transposon system of the photosynthetic cyanobacterium Scytonema hofmanni. The Cas12k complex structure reveals an intricate guide RNA architecture and critical interactions mediating RNA-guided target DNA recognition. TnsC helical filament assembly is ATP-dependent and accompanied by structural remodelling of the bound DNA duplex. In vivo transposition assays corroborate key features of the structures, and biochemical experiments show that TniQ restricts TnsC polymerization, while TnsB interacts directly with TnsC filaments to trigger their disassembly upon ATP hydrolysis. Together, these results suggest that RNA-directed target selection by Cas12k primes TnsC polymerization and DNA remodelling, generating a recruitment platform for TnsB to catalyse site-specific transposon insertion. Insights from this work will inform the development of CRISPR-associated transposons as programmable site-specific gene insertion tools.
History
DepositionAug 30, 2021-
Header (metadata) releaseDec 1, 2021-
Map releaseDec 1, 2021-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7pla
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13486.png

Downloads & links

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Map

FileDownload / File: emd_13486.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.64 Å
Density
Contour LevelBy AUTHOR: 0.055 / Movie #1: 0.06
Minimum - Maximum-0.09044516 - 0.29100224
Average (Standard dev.)0.005274707 (±0.013449504)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 192.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.640.640.64
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z192.000192.000192.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S213
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0900.2910.005

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Supplemental data

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Sample components

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Entire : Ternary complex of ShCas12k with sgRNA and target dsDNA

EntireName: Ternary complex of ShCas12k with sgRNA and target dsDNA
Components
  • Complex: Ternary complex of ShCas12k with sgRNA and target dsDNA
    • Protein or peptide: ShCas12k
    • RNA: sgRNA
    • DNA: DNA target strand
    • DNA: DNA non-target strand

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Supramolecule #1: Ternary complex of ShCas12k with sgRNA and target dsDNA

SupramoleculeName: Ternary complex of ShCas12k with sgRNA and target dsDNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 187 KDa

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Macromolecule #1: ShCas12k

MacromoleculeName: ShCas12k / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 73.421773 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SNASQITIQA RLISFESNRQ QLWKLMADLN TPLINELLCQ LGQHPDFEKW QQKGKLPSTV VSQLCQPLKT DPRFAGQPSR LYMSAIHIV DYIYKSWLAI QKRLQQQLDG KTRWLEMLNS DAELVELSGD TLEAIRVKAA EILAIAMPAS ESDSASPKGK K GKKEKKPS ...String:
SNASQITIQA RLISFESNRQ QLWKLMADLN TPLINELLCQ LGQHPDFEKW QQKGKLPSTV VSQLCQPLKT DPRFAGQPSR LYMSAIHIV DYIYKSWLAI QKRLQQQLDG KTRWLEMLNS DAELVELSGD TLEAIRVKAA EILAIAMPAS ESDSASPKGK K GKKEKKPS SSSPKRSLSK TLFDAYQETE DIKSRSAISY LLKNGCKLTD KEEDSEKFAK RRRQVEIQIQ RLTEKLISRM PK GRDLTNA KWLETLLTAT TTVAEDNAQA KRWQDILLTR SSSLPFPLVF ETNEDMVWSK NQKGRLCVHF NGLSDLIFEV YCG NRQLHW FQRFLEDQQT KRKSKNQHSS GLFTLRNGHL VWLEGEGKGE PWNLHHLTLY CCVDNRLWTE EGTEIVRQEK ADEI TKFIT NMKKKSDLSD TQQALIQRKQ STLTRINNSF ERPSQPLYQG QSHILVGVSL GLEKPATVAV VDAIANKVLA YRSIK QLLG DNYELLNRQR RQQQYLSHER HKAQKNFSPN QFGASELGQH IDRLLAKAIV ALARTYKAGS IVLPKLGDMR EVVQSE IQA IAEQKFPGYI EGQQKYAKQY RVNVHRWSYG RLIQSIQSKA AQTGIVIEEG KQPIRGSPHD KAKELALSAY NLRLTRR S

UniProtKB: ShCas12k

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Macromolecule #2: sgRNA

MacromoleculeName: sgRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Scytonema hofmannii (bacteria)
Molecular weightTheoretical: 82.376547 KDa
SequenceString: GGAUAUUAAU AGCGCCGCAA UUCAUGCUGC UUGCAGCCUC UGAAUUUUGU UAAAUGAGGG UUAGUUUGAC UGUAUAAAUA CAGUCUUGC UUUCUGACCC UGGUAGCUGC UCACCCUGAU GCUGCUGUCA AUAGACAGGA UAGGUGCGCU CCCAGCAAUA A GGGCGCGG ...String:
GGAUAUUAAU AGCGCCGCAA UUCAUGCUGC UUGCAGCCUC UGAAUUUUGU UAAAUGAGGG UUAGUUUGAC UGUAUAAAUA CAGUCUUGC UUUCUGACCC UGGUAGCUGC UCACCCUGAU GCUGCUGUCA AUAGACAGGA UAGGUGCGCU CCCAGCAAUA A GGGCGCGG AUGUACUGCU GUAGUGGCUA CUGAAUCACC CCCGAUCAAG GGGGAACCCU AAAUGGGUUG AAAGGAGAAG UC AUUUAAU AAGGCCACU

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Macromolecule #3: DNA target strand

MacromoleculeName: DNA target strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.301837 KDa
SequenceString:
(DG)(DT)(DT)(DT)(DA)(DA)(DC)(DA)(DG)(DT) (DG)(DG)(DC)(DC)(DT)(DT)(DA)(DT)(DT)(DA) (DA)(DA)(DT)(DG)(DA)(DC)(DT)(DT)(DC) (DT)(DC)(DA)(DA)(DC)(DC)(DT)(DC)(DC)(DT) (DA) (DC)(DG)(DT)(DA)(DT)(DC)(DG)(DT) (DA)(DG)

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Macromolecule #4: DNA non-target strand

MacromoleculeName: DNA non-target strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.497987 KDa
SequenceString:
(DC)(DT)(DA)(DC)(DG)(DA)(DT)(DA)(DC)(DG) (DT)(DA)(DG)(DG)(DA)(DG)(DG)(DT)(DT)(DG) (DA)(DG)(DA)(DA)(DG)(DT)(DC)(DA)(DT) (DT)(DT)(DA)(DA)(DT)(DA)(DA)(DG)(DG)(DC) (DC) (DA)(DC)(DT)(DG)(DT)(DT)(DA)(DA) (DA)(DC)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 51.81 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 138000
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER

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